Roberta Asci

Homozygous mutation in the prokineticin-receptor2 gene (Val274Asp) presenting as reversible Kallmann syndrome and persistent oligozoospermia: Case Report

Prokineticin 2 (Prok2) or prokineticin-receptor2 (Prok-R2) gene mutations are associated with Kallmann syndrome (KS). We describe a new homozygous mutation of Prok-R2 gene in a man displaying KS with an apparent reversal of hypogonadism. The proband, offspring of consanguineous parents, presented at age 19 years with absent puberty, no sense of smell, low testosterone and gonadotrophin levels. Magnetic resonance imaging showed olfactory bulb absence. The patient achieved virilization and spermatogenesis with gonadotrophin administration. Two years after discontinuing hormonal therapy, he maintained moderate oligozoospermia and normal testosterone levels. Prok2 and Prok-R2 gene sequence analyses were performed. The proband had a homozygous mutation in Prok-R2 exon 2 that harbours the c.T820>A base substitution, causing the introduction of an aspartic acid in place of valine at position 274 (Val274Asp). His mother had the same mutation in heterozygous state. This report describes a novel homozygous mutation of Prok-R2 gene in a man with variant KS, underlying the role of Prok-R2 gene in the olfactory and reproductive system development in humans. Present findings indicate that markedly delayed activation of gonadotrophin secretion may occur in some KS cases with definite gene defects, and that oligozoospermia might result from a variant form of reversible hypogonadotrophic hypogonadism.

Regulation of divalent metal transporter 1 (DMT1) non-IRE isoform by the microRNA Let-7d in erythroid cells

Background Divalent metal transporter 1 (DMT1) is a widely expressed metal-iron transporter gene encoding four variant mRNA transcripts, differing for alternative promoter at 5′ (DMT1 1A and 1B) and alternative splicing at 3′ UTR, differing by a specific sequence either containing or lacking an iron regulatory element (+IRE and -IRE, respectively). DMT1-IRE might be the major DMT1 isoform expressed in erythroid cells, although its regulation pathways are still unknown. Design and Methods The microRNA (miRNA) Let-7d (miR-Let-7d) was selected by the analysis of four miRNAs, predicted to target the DMT1-IRE gene in CD34+ hematopoietic progenitor cells, in K562 and in HEL cells induced to erythroid differentiation. Using a luciferase reporter assay we demonstrated the inhibition of DMT1-IRE by miR-Let-7d in K562 and HEL cells. The function of miR-Let-7d in erythroid cells was evaluated by the flow cytometry analysis of erythroid differentiation markers, by benzidine staining and by iron flame atomic absorption for the evaluation of iron concentration in the endosomes from K562 cells over-expressing miR-Let-7d. Results We show that in erythroid cells, DMT1-IRE expression is under the regulation of miR-Let-7d. DMT1-IRE and miR-Let-7d are inversely correlated with CD34+ cells, K562 and HEL cells during erythroid differentiation. Moreover, overexpression of miR-Let-7d decreases the expression of DMT1-IRE at the mRNA and protein levels in K562 and HEL cells. MiR-Let-7d impairs erythroid differentiation of K562 cells by accumulation of iron in the endosomes. Conclusions Overall, these data suggest that miR-Let-7d participates in the finely tuned regulation of iron metabolism by targeting DMT1-IRE isoform in erythroid cells.

Molecular analysis of 42 patients with congenital dyserythropoietic anemia type II: new mutations in the SEC23B gene and a search for a genotype-phenotype relationship

Background The most frequent form of congenital dyserythropoietic anemia is the type II form. Recently it was shown that the vast majority of patients with congenital dyserythropoietic anemia type II carry mutations in the SEC23B gene. Here we established the molecular basis of 42 cases of congenital dyserythropoietic anemia type II and attempted to define a genotype-phenotype relationship. Design and Methods SEC23B gene sequencing analysis was performed to assess the diversity and incidence of each mutation in 42 patients with congenital dyserythropoietic anemia type II (25 described exclusively in this work), from the Italian and the French Registries, and the relationship of these mutations with the clinical presentation. To this purpose, we divided the patients into two groups: (i) patients with two missense mutations and (ii) patients with one nonsense and one missense mutation. Results We found 22 mutations of uneven frequency, including seven novel mutations. Compound heterozygosity for a missense and a nonsense mutation tended to produce a more severe clinical presentation, a lower reticulocyte count, a higher serum ferritin level, and, in some cases, more pronounced transfusion needs, than homozygosity or compound heterozygosity for two missense mutations. Homozygosity or compound heterozygosity for two nonsense mutations was never found. Conclusions This study allowed us to determine the most frequent mutations in patients with congenital dyserythropoietic anemia type II. Correlations between the mutations and various biological parameters suggested that the association of one missense mutation and one nonsense mutation was significantly more deleterious that the association of two missense mutations. However, there was an overlap between the two categories.

New understandings of the genetic basis of isolated idiopathic central hypogonadism

Idiopathic hypogonadotropic hypogonadism is a rare disease that is characterized by delayed/absent puberty and/or infertility due to an insufficient stimulation of an otherwise normal pituitary-gonadal axis by gonadotrophin-releasing hormone (GnRH) action. Because reduced or normal luteinizing hormone (LH)/follicle-stimulating hormone (FSH) levels may be observed in the affected patients, the term idiopathic central hypogonadism (ICH) appears to be more appropriate. This disease should be distinguished from central hypogonadism that is combined with other pituitary deficiencies. Isolated ICH has a complex pathogenesis and is fivefold more prevalent in males. ICH frequently appears in a sporadic form, but several familial cases have also been reported. This finding, in conjunction with the description of numerous pathogenetic gene variants and the generation of several knockout models, supports the existence of a strong genetic component. ICH may be associated with several morphogenetic abnormalities, which include osmic defects that, with ICH, constitute the cardinal manifestations of Kallmann syndrome (KS). KS accounts for approximately 40% of the total ICH cases and has been generally considered to be a distinct subgroup. However, the description of several pedigrees, which include relatives who are affected either with isolated osmic defects, KS, or normo-osmic ICH (nICH), justifies the emerging idea that ICH is a complex genetic disease that is characterized by variable expressivity and penetrance. In this context, either multiple gene variants or environmental factors and epigenetic modifications may contribute to the variable disease manifestations. We review the genetic mechanisms that are presently known to be involved in ICH pathogenesis and provide a clinical overview of the 227 cases that have been collected by the collaborating centres of the Italian ICH Network.

Missense mutations in the ABCB6 transporter cause dominant familialpseudohyperkalemia

Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by increased serum [K+] in whole blood stored at or below room temperature, without additional hematological abnormalities. Functional gene mapping and sequencing analysis of the candidate genes within the 2q35–q36 critical interval identified—in 20 affected individuals among three multigenerational FP families—two novel heterozygous missense mutations in the ABCB6 gene that cosegregated with disease phenotype. The two genomic substitutions altered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that, in erythrocyte membranes, bears the Langereis blood group antigen system. The ABCB6 R375Q mutation did not alter the levels of mRNA or protein, or protein localization in mature erythrocytes or erythroid precursor cells, but it is predicted to modestly alter protein structure. ABCB6 mRNA and protein levels increase during in vitro erythroid differentiation of CD34+ erythroid precursors and the erythroleukemia cell lines HEL and K562. These data suggest that the two missense mutations in residue 375 of the ABCB6 polypeptide found in affected individuals of families with chromosome 2‐linked FP could contribute to the red cell K+ leak characteristic of this condition. Am. J. Hematol. 2013.

Mutational spectrum in congenital dyserythropoietic anemia type II: Identification of 19 novel variants in SEC23B gene

SEC23B gene encodes an essential component of the coat protein complex II (COPII)‐coated vesicles. Mutations in this gene cause the vast majority the congenital dyserythropoietic anemia Type II (CDA II), a rare disorder resulting from impaired erythropoiesis. Here, we investigated 28 CDA II patients from 21 unrelated families enrolled in the CDA II International Registry. Overall, we found 19 novel variants [c.2270 A>C p.H757P; c.2149−2 A>G; c.1109+1 G>A; c.387(delG) p.L129LfsX26; c.1858 A>G p.M620V; c.1832 G>C p.R611P; c.1735 T>A p.Y579N; c.1254 T>G p.I418M; c.1015 C>T p.R339X; c.1603 C>T p.R535X; c.1654 C>T p.L552F; c.1307 C>T p.S436L; c.279+3 A>G; c. 2150(delC) p.A717VfsX7; c.1733 T>C p.L578P; c.1109+5 G>A; c.221+31 A>G; c.367 C>T p.R123X; c.1857_1859delCAT; p.I619del] in the homozygous or the compound heterozygous state. Homozygosity or compound heterozygosity for two nonsense mutations was never found. In four cases the sequencing analysis has failed to find two mutations. To discuss the putative functional consequences of missense mutations, computational analysis and sequence alignment were performed. Our data underscore the high allelic heterogeneity of CDA II, as the most of SEC23B variations are inherited as private mutations. In this mutation update, we also provided a tool to improve and facilitate the molecular diagnosis of CDA II by defining the frequency of mutations in each exon. Am. J. Hematol., 2010.

Trasferrin receptor 2 gene regulation by microRNA 221 in SH-SY5Y cells treated with MPP⁺ as Parkinson's disease cellular model.

Parkinsons disease (PD) is one of the most frequent human neurodegenerations. The neurodegeneration in PD is related to cellular iron increase but the mechanisms involved in iron accumulation remain unclear. Transferrin receptor type 2 (TFR2) is a protein expressed on cell membrane and involved in the cellular iron uptake. We hypothesized that microRNA 221 could regulate the expression of TfR2 in an in vitro model of Parkinsons disease, SH-SY5Y cells treated with MPP⁺. The miRNA 221 was selected by in silico analysis of several miRNAs predicted to target the TFR2 gene in SHSY5Y cells treated with MPP⁺. Taqman miRNA assay was used to evaluate the expression of the selected miRNAs. Using a luciferase assay we demonstrated the inhibition of TFR2 by miRNA 221. We show that in PD cellular model, TFR2 expression is regulated by miRNA 221. TFR2 and miR 221 are inversely correlated in SHSY5Y cells during the treatment with MPP⁺. Moreover, overexpression of miRNA 221 decreases the expression of TFR2, respectively, at the mRNA and protein levels. The inhibition of endogenous miRNA 221 also is able to regulate TFR2. These data suggest that miRNA 221 regulate TFR2 in PD model.

Congenital dyserythropoietic anaemias: new acquisitions

Dyserythropoiesis is defined as a condition of abnormal erythropoiesis in which there are both morphological and functional disorders, with the predominant phenomena being erythroblast abnormalities and ineffective erythropoiesis, respectively. Dyserythropoietic anaemias can be divided into primary and secondary forms and both inherited and acquired forms occur. The congenital dyserythropoietic anaemias (CDA) have been classified into three types (CDA I, II and III) in relation to their pattern of inheritance and bone marrow morphology. There is also a huge group of congenital forms that cannot be included with any of the three canonical types and remain difficult to diagnose (CDA IV-VII)1.

Diagnosi e terapia dell’ipogonadismo nella sindrome di Kallmann

RiassuntoLa sindrome di Kallmann (SK) è una forma congenita di ipogonadismo ipogonadotropo idiopatico in cui l’ipogonadismo si associa a deficit olfattivo (ipo/anosmia) e ad altri difetti non riproduttivi (agenesia renale, sincinesie, palatoschisi, sindattilia). La SK può essere sporadica o familiare, con ereditarietà X-linked, autosomica (dominante o recessiva) o oligogenica. Nel 30% dei casi è stata trovata una mutazione di geni che regolano la migrazione dei neuroni GnRH e/o la morfogenesi dei bulbi olfattori (KAL1, FGFR1, FGF8, PROKR2, PROK2, CHD7). La SK si manifesta come ritardo o assenza della pubertà dopo i 18 anni nel maschio ed i 16 nella donna, insieme con il deficit olfattivo e, incostantemente, con le anomalie somatiche. I bassi livelli di gonadotropine e testosterone (T) o estradiolo (E2) sono diagnostici di ipogonadismo solo dopo l’eta’ di 18 (nei maschi) e 16 anni (nelle donne) e tra la seconda settimana e il terzo mese di vita nei bambini con micropene e/o criptorchidismo. In età adolescenziale i test ormonali basali e dopo stimolo non permettono di differenziare il ritardo costituzionale della pubertà dall’ipogonadismo ipogonadotropo. Lo smell test e la risonanza magnetica sono utili per valutare il deficit sensoriale e la morfologia dei bulbi olfattivi. Il sequenziamento dei geni candidati va effettuato sulla base dei dati clinici e dell’ereditarietà. Per indurre la pubertà si somministrano dosi crescenti di T intramuscolo (i.m.) o transdermico (td) nell’uomo o di E2 (orale, td) nella donna. Nei ragazzi può essere stimolato il T endogeno con l’hCG i.m. L’incremento delle dosi sostitutive va effettuato ogni 3–6 mesi sulla base della risposta clinica e del monitoraggio di laboratorio. Dopo la pubertà la terapia può essere continuata con il T undecaonato i.m. (ogni 10–12 settimane) nel maschio e con combinazioni di estro-progestinici nella donna. Un periodo di sospensione va programmato dopo il completamento della pubertà per poter identificare le forme reversibili. La terapia con gonadotropine (hCG ed FSH nell’uomo, FSH seguito da hCG nella donna) stimola la gametogenesi ed è indicata se si desidera la fertilità. Il counseling genetico permette di determinare il rischio potenziale di trasmissione della sindrome.